The stable propagation of information from one generation to the next is one of the of the most basic processes required for life. Because this information is encoded in the DNA, the molecular mechanisms regulating replication of DNA are fundamental to life itself. These mechanisms ensure that replication occurs at the proper time during the cell cycle, that the genome is replicated accurately, and that the entire genome is replicated only once in each cell cycle. In eukaryotes, the proteins which coordinate these three essential processes are still not well understood. Much of the regulation of DNA replication centers around the events which control initiation. There is now overwhelming biochemical evidence which demonstrates that initiation in both prokaryotic and eukaryotic organisms involves the interaction between specific DNA sequences which act as sites for initiation (origins of replication), and specific proteins which recognize these origin sites (origin recognition complexes, ORC). Further, visual observation of the replication process inside the nuclei of higher eukaryotic cells, strongly suggests that the clustered organization of multiple loops of chromatin around a central hub or "foci" plays a key role in regulating initiation of replication. Over the last few years we have used a simple cell-free system derived from Xenopus eggs, which efficiently re-creates the replication process occurring inside nuclei to characterize the organization of chromatin around these foci and to identify proteins essential to their formation. Our results support a model in which multiple loops of chromatin aggregate around 200-300 discrete foci structures before, during, and after DNA replication. That these foci are important for regulating the initiation of replication is supported by the finding that the replication protein RP-A, binds specifically to foci prior to initiation, and that DNA at foci is labeled during initiation. Importantly, to better understand the biochemical composition of foci we have developed assays for purifying loci components and have successfully used these assays to purify, to homogeneity, a novel protein essential for formation of foci, FFA-1. In the coming grant period we will use this readily manipulatable in vitro system to further characterize athe role that foci structure and foci proteins play in DNA replication. Specifically, we will determine how FFA- 1 contributes to the initiation of replication at foci. 2. We will isolate and characterize two other proteins essential for formation of foci. 3. We will determine whether replicon size and origin selection are regulated by foci size. 4. We will determine whether the organization of chromatin around foci is stable from one S-phase to the next. 5. We will use this system to clone and characterize potential DNA sequence with high affinity for foci sites and/or that are selectively used as origins. Successful completion of this work will provide a foundation for understanding how foci form and what role this subnuclear compartment plays in ensuring that DNA replication occurs successfully.